Conventional commercially-available embryo culture media for human ART procedures contain human serum albumin (HSA) obtained from human blood and tissue sources. In some laboratories bovine serum albumin (BSA) is also used as a source of protein in human embryo culture procedures (Loutradis et al., 1992; Quinn, 1994), similarly obtained from blood and tissue sources from cows. The efficiency of media containing HSA and BSA was reported to be similar (Staessen et al., 1998). The use in culture medium of protein obtained from donors (human or bovine) has the potential to transmit diseases to patients undergoing assisted conception treatment. The generation of viable human embryos in a chemically-defined culture system (devoid of added protein or albumin, or animal or human-derived protein extract) beginning from oocyte collection, followed by sperm washing, insemination, fertilization up to the cleaved embryo stage and finally embryo transfer has not been described, although chemically-defined media for mouse, rabbit and primates have been reported in recent years (Spindle, 1995; Li et al., 1996; Schramm and Bavister, 1996). Previous claims of a chemically defined protein-free medium (PFM) for human application, in fact, were not truly protein-free, because the sperm meant for fertilization was still prepared in medium that contained serum proteins (Mohr & Trounson, 1986; Serta and Kiessling 1997 (Abst); Parinaud et al. 1999). A totally protein-free media for ART procedures thus has not been described hitherto.
The availability of a chemically defined medium used for generating viable early cleavage stage human embryos is needed in the art to ensure the safety of patients undergoing assisted reproduction treatment by avoiding the use of potentially hazardous donor protein. Concern over possible transmission of pathogenic diseases, in particular viral diseases, such as human acquired immunodeficiency disease syndrome (AIDS) and hepatitis, or Creutzfeldt-Jakob disease (CJD) transmitted by prions or others in blood-derived products has led a number of providers of healthcare services in the area of ART worldwide to seek alternative(s) to donor protein for their embryo culture and handling procedures.
The transmission of a deadly viral disease (AIDS) to hemophiliacs through blood-derived products is well documented (See, for example, Craven et al., 1997, Med. Sci. Law 37: 215-227; Keshavjee et al., 2001, Soc. Sci. Med. 53: 1081-1094; Weinberg et al., 2002, Ann. Intern. Med. 136: 312-319; Evatt, 2006, Semin. Hematol. 43: S4-9). Human growth hormone extracted from the pituitary was found to be capable of transmitting CJD to humans (Esmonde et al., 1994) and human gonadotropin injections could also transmit CJD from person to person (CDC, 1985). CJD can be transmitted through blood (although the titer of CJD prions is low in blood; Heye et al., 1994). In the past an epidemic of hepatitis B occurred in about 200 IVF patients that received embryos cultured in medium containing pooled sera contaminated with hepatitis B virus (van Os et al., 1991). Recently the scientific community was confronted with the dilemma of having to inform their patients that a commercial preparation of a culture medium used for embryo culture and handling may be contaminated with albumin donated by a person who later died of CJD (Kemmann, 1998).
There are a number of reports of development of mouse blastocysts from zygote and cleaved stages in a protein-free medium. The earliest reports include those of Brinster (1965) and, Cholewa and Whitten (1970) who used polyvinylpyrrolidone (PVP), which is a high molecular weight colloid, as a lubricant and for increasing the viscosity of the medium. Subsequent to this work a number of other workers have successfully cultured mouse as well as other mammalian embryos in protein-free media (Dandekar and Glass, 1990; Spindle, 1995; Li et al., 1996; Schramm and Bavister, 1996). In recent years, in addition to PVP, polyvinyl alcohol (PVA) (Bavister, 1995) has also been used to replace serum protein in culture medium. For example, Biggers et al. (1997) investigated the effect of replacing bovine serum albumin (BSA) with polyvinyl alcohol (PVA) and/or amino acids on mouse zygote development. They observed that PVA could not be substituted completely for BSA in the mouse embryo culture medium. The effect of PVA on rate of blastocyst development was only slightly less than with BSA but the rate of partial hatching was significantly less. Substitution of BSA with PVA lowered the overall response but did not lead to major perturbations.
In addition to its many biological roles, serum proteins confer useful physical attributes such as lubrication and viscosity in the culture medium. Increased viscosity and lubrication in the culture medium is required for ease of handling and manipulation of the embryo and to prevent it from adhering to the walls of the culture dish and embryo transfer catheters. The incorporation of PVP and PVA merely serve to duplicate the physical attributes of serum proteins. However, PVP and PVA are not sources of fixed nitrogen and they do not perform the various biological roles of protein. In addition, the teratological properties of PVP and PVA have not been fully examined, which make their use in human therapeutic assisted reproduction questionable (Gardner and Lane, 1998a).
Some investigators have attempted to generate viable human embryos in a protein-free culture system, most recently Serta et al. (1997) and Parinaud et al. (1998a). However Serta and co-workers (1997) prepared their spermatozoa for insemination by swim-down through a column of BSA (although subsequent culture was performed in a protein-free medium). These workers achieved a pregnancy rate of 31% (n=45) some of which have proceeded to term with the birth of normal offspring. Parinaud et al. (1998a) obtained fertilizations with spermatozoa prepared in a protein-free medium when insemination was performed in the same medium. However the resultant zygotes were cultured in BM1 medium, and although this reference did not specify whether their BM1 medium contained protein, a previous publication from the same group suggested BM1 medium contained 1% HSA (Parinaud et al., 1998b).
Some workers have shown that replacement of serum protein with a single antioxidant and chelator such as EDTA (Mehta and Kiessling, 1990; Serta et al., 1997) does not impair fertilization and cleavage of viable embryos in the mouse and human. However Serta et al. (1997) suggested that the embryo transfer catheter be rinsed extensively with the protein-free medium, which implies the tendency of embryos to stick to the inner wall of embryo transfer catheter. Serum protein also has a role in maintaining pH in culture medium (Moessner et al., 1993). Beside its role as a nutrient in biological systems, protein has a number of other roles such as a chain-breaking antioxidant and a chelator of metal ions (Barber, 1961; Vidlakova et al., 1972; Wayner et al., 1987).
The physiological functions of albumin and plasma proteins in general are well documented. The role of albumin in preventing membrane peroxidation indicates a direct role in membrane stability. It is involved in capillary membrane permeability and in osmoregulation. Albumin provides 80% of the total colloid osmotic pressure in plasma. Albumin is involved in the transport of carbon dioxide and acts as a pH buffer; albumin accounts for the greatest (95%) portion of the non-bicarbonate buffer value of plasma. Proteins also serve as a source of energy. Deaminated alanine is pyruvate, which can be either converted to acetyl-CoA or glucose and glycogen. Albumin may help solubilize lipids and transports hormones, vitamins and metals. It serves as reservoirs for the release and use of these components.
Any attempt at substituting serum albumin in culture medium should therefore take into consideration these in vivo roles and physical attributes which are useful for embryo handling and manipulation in vitro. A single component may not fulfill all the functions of serum protein.
Although protein-free media that supports development of a number of animal species has been described previously, no such protein-free media has been successfully used in humans, nor could such media be presumed to support or be optimal for human embryo development. Thus there exists a distinct need for a defined, protein-free growth medium especially adapted for human ART and IVF.